First detection of hepatitis E virus in dromedary camels from Iran

Abstract Background Hepatitis E virus (HEV) genotype 7 is a zoonotic disease detected in dromedary camels. Hypothesis/objectives The consumption of camel meat and dairy products, the abundance of dromedary camels in Southeast Iran and the import of camels from neighbouring countries to Iran made the researchers investigate the infection rate of camels by the virus. Animals A total of 53 healthy camels in Southeast Iran (Sistan and Baluchistan Province) tested for HEV RNA. Method A total of 17 blood samples and 36 liver samples were taken from 53 healthy dromedary camels (aged between 2 and 10 years) from various southeastern regions of Iran. The samples were tested for HEV using RT‐PCR. Results Overall, 56.6% of the studied samples (n = 30) tested positive for HEV RNA. Conclusions and clinical importance The present study was the first of its kind in Iran and revealed the presence of HEV in the Iranian dromedary camel population, which might play the role of a zoonosis reservoir for its transmission to humans. This discovery raises concerns about food‐borne illnesses that can be transmitted from animals to humans. However, further research is needed to identify the specific genotype of the HEV in Iranian dromedary camel infections and to determine the risk of spread to other animals and humans.

hepatitis after infection with HEV (Bari et al., 2021;Meng, 2010;Woo et al., 2014Woo et al., , 2016. The mortality rate caused by the HEV infection is 1%-2% in the general population; however, it increases up to 10%-25% in pregnant women and above 70% in patients suffering from liver damages (Taherkhani & Farshadpour, 2016). The HEV strains remain silent and subclinical in most animal species and represent no clinical symptoms; however, the infected animals might indicate microscopic hepatitis evidence (Meng, 2010).
According to the genomic sequence analysis, the HEV strains from humans and other mammals are categorized into eight genotypes. The strains of HEV genotypes 1 and 2 only infect humans, are endemic in developing countries and are primarily transmitted via the faecaloral route. In contrast, strains from genotypes 3 and 4 have a broader host range and geographical spread and circulate among humans, wild boars, pigs, deer, mongoose, macaques, sheep, cows, yaks and rabbits, which are the sporadic agents of human hepatitis E in underdeveloped and industrial countries (Aggarwal, 2011). These strains induce silent infections in various mammals and are sometimes transmitted to humans. HEV-5 and HEV-6 are the new genotypes detected in Japanese wild boars. The HEV-7 and HEV-8 genotypes have also recently been detected in dromedary and Bactrian camels, respectively (Sridhar et al., 2017). HEV was first identified in dromedary camels in the Middle East in 2014 and was classified in genus Orthohepevirus A under dromedary Camel HEV (DcHEV) (Woo et al., 2014). The transmission of HEV-7 to nonhuman primates and to a person in the UAE who consumed infected camel products confirmed its zoonotic nature (Lee et al., 2016;Li et al., 2016). Furthermore, a long-term epidemiological study revealed that dromedary camels have long suffered from DcHEV and that the virus is geographically extensive and diverse (Rasche et al., 2016). Hepatitis E is endemic in many Middle Eastern countries such as Iran; however, the presence of HEV RNA has not been addressed in previous studies. Accordingly, the distribution pattern of the HEV genotypes is still unknown in Iran (Khuroo et al., 2016;Taherkhani & Farshadpour, 2016), and the zoonotic role of animals has yet to be investigated in this regard. The increase in camel meat and dairy consumption, the abundance of dromedary camels in Southeast Iran and the import of camels from Pakistan and Afghanistan to these regions highlight the need for studies examining the infection rate of the virus among dromedary camels through reverse transcription polymerase chain reaction (RT-PCR). The present study was the first of its kind in Iran. liver and blood, the RNA purification was carried out using the DNase (ZandBiotech, Co., Iran) commercial kit according to a kit protocol to eliminate possible DNA contaminations. The purified RNAs were then used for cDNA synthesis using the Pars Toos commercial kit.

MATERIALS AND METHODS
All stages, from RNA extraction to cDNA synthesis, were carried out immediately after sampling on the same day. The extracted samples were stored in freezers at −80 • C for the subsequent examinations.
The Actin Beta-like2 gene (ACTBL2) was used as a reference gene to ensure high-quality cDNA synthesis. The gene was found to be 100% present in dromedary camels after blast analysis. After alignment and thermodynamic evaluation of the primers, the forward and reverse gene reference primers 5′-TATTGGCAACGAGCGGTTCC-3′ and 5′-GGCATAGAGGTCTTTACGGATGTC-3′, respectively, were selected. An ordinary PCR was performed with the reference gene primers and synthesized cDNAs at 54 • C to verify cDNA quality. was obtained from the Department of Microbiology, which was, in fact, a synthesized HEV sample with primers explicitly used for HEV (Hajiahmadi et al., 2017). The PCR products were loaded on a 2.5% agarose, run at 70 V for 70 min and imaged using a gel documentation system. PCR was repeated, and the results were recorded to confirm the positive samples (Figures 2 and 3). The data were then imported to SPSS software version 28 and analysed using the Fisher exact test.

RESULTS
Out of the 53 liver and blood samples obtained from apparently healthy camels in Southeast Iran, 56.6% (n = 30) tested positive for HEV RNA using the RT-PCR method. Statistical data analysis using the Fisher exact test indicated a significant relationship between the camels' hep-atitis E infection and their geographical location (p < 0.001) so that the infection rate was higher in camels from Mirjaveh County than in those from other counties. The results also indicated the high frequency of infection in camels imported from Pakistan and Afghanistan. However, no serum sample tested positive for HEV RNA (Bassal et al., 2019). A study in 2020 revealed that the prevalence of DcHEV-Abs in dromedary camel serum samples collected from Saudi Arabia was 23.1% (El-Kafrawy et al., 2020). Another study in 2021 found that 2.2% of stool samples taken from dromedary camels in Ethiopia (one case out of 45 samples) tested positive for HEV RNA using the nested reverse transcription polymerase chain reaction. method (Bari et al., 2021). between gender and HEV infection (Li et al., 2017;Ouoba et al., 2019).
Therefore, further studies on risk factors are required in this regard.
Our study found a significant relationship between the distribution of camels' hepatitis E infection and their age group so that the infection rate was significantly higher in camels under 2 years of age compared to other ages (p < 0.001). According to Corman et al., 2020, the natural process of the Genotype HEV-7 infection in camels is similar to that of HEV-3 in pigs in their first 6 months of life, which is associated with a decline in maternal antibody levels. This is consistent with the present study regarding the parameter of age. The study of El-Kafrawy et al. in 2022 also reported the highest prevalence of HEV infection in young, single-humped camels under 1 year of age (1/8, 12.5%), followed by the age group of 1-3 years (9/229, 3.9%), which is consistent with our study findings. Moreover, a recent study in the UAE showed that all camels were infected in the first 6 months of life and cleared the virus after an average of 2 months (Corman et al., 2020). As the natural course of HEV-7 genotype infection in camels usually occurs in the first 6 months of life, the viremia period lasts on average 8 weeks (Corman et al., 2020), and camels are usually slaughtered at higher age (2 years old even in industrial farming) (Kadim et al., 2013), their meat products may have a less risky than pigs in terms of HEV transmission (Corman et al., 2020).
The present study was the first of its kind in Iran and revealed the presence of HEV in the Iranian dromedary camel population, which might play the role of a zoonosis reservoir for its transmission to humans. According to the religious consideration in Iran, boars and pigs are not bred and consumed in Iran and thus do not count as the main hepatitis E reservoirs in the country; as such, the role of other animals in the transmission of this infection must be investigated. More specifically, the consumption of camel milk and meat in the country and the reports of HEV transmission to humans via the consumption of camel products in the UAE indicate that the role of DcHEV in the human population of Iran must be specified. As HEV has been detected in the stool samples of camels (Bari et al., 2021;Rasche et al., 2016;Woo et al., 2014), camels can play a role in transmitting this virus to other animals and cause environmental problems. Accordingly, other wild or domestic animals in close contact with dromedary camels must be investigated to evaluate the HEV-7 host range (Ouoba et al., 2019). The limitation of the present study is that the detection of anti-DcHEV antibody from the collected samples was not carried out simultaneously to present the actual HEV infection rate, which must be considered in future studies. In this regard, the study of the HEV prevalence in the Iranian camel population and clarification of the disease load, the identification of the HEV genotype involved in dromedary camel infection and the evaluation of its zoonosis potential and transmission to other animals require further investigation. Moreover, further investigation should suggest gene sequencing and sequence analysis for detailed molecular analysis. Moreover, serological and molecular studies of the virus are recommended in other animal species and their various samples.